The present invention relates to 5-thia-xcfx89-substituted phenylprostaglandin E derivatives.
More particularly, it relates to 5-thia-xcfx89-substituted phenylprostaglandin E derivatives of the formula (I) 
(wherein, ail the symbols are as defined hereafter).
Prostaglandin E2 (abbreviated as PGE2) has been known as a metabolite in the arachidonate cascade. It has been known that PGE2 possesses cyto-protective activity, uterine contractile activity, a pain-inducing effect, a promoting effect on digestive peristalsis, an awakening effect, a suppressive effect on gastric acid secretion, hypotensive activity and diuretic activity etc.
In the recent study, it was found that PGE2 receptor was divided into some subtypes which possess different physical role from each other. At present, four receptor subtypes are known and they are called EP1, EP2, EP3, EP4 (Negishi M. et al, J. Lipid Mediators Cell Signaling, 12, 379-391 (1995)).
The present inventors have studied to find the compound which can bind to each receptor specifically, have found that the compound of the present invention can bind to EP4 subtype receptor strongly, and then have achieved the present invention.
It is thought that EP4 subtype receptor relates to inhibition of producing TNF-xcex1 and acceleration of producing IL-10. Therefore, the compounds of the present invention which can bind EP4 subtype receptor strongly are expected to be useful for the prevention and/or treatment of immunological diseases (autoimmune diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis, Sjoegren""s syndrome, chronic rheumarthrosis and systemic lupus erythematosus etc., and rejection after organ transplantation etc.), asthma, abnormal bone formation, neuronal cell death, lung failure, liver damage, acute hepatitis, nephritis, renal insufficiency, hypertension, myocardiac ischemia, systemic inflammatory response syndrome, ambustion pain, sepsis, hemophagous syndrome, macrophage activation syndrome, Still""s disease, Kawasaki disease, burn, systemic granulomatosis, ulcerative colitis, Crohn""s disease, hypercytokinemia at dialysis, multiple organ failure, and shock etc.
Further, it is thought that EP4 subtype receptor relates to sleeping disorder and blood platelet aggregation, so the compounds of the present invention are expected to be useful for the prevention and/or treatment of such diseases.
The compounds of the present invention of the formula (I) bind weakly to the other subtype receptors and do not express other effects, so such compounds are expected to be an agent having less side effect.
On the other hand, a lot of prostaglandins in which carbon atom at 5th position in PG skeleton was replaced with sulfur atom and which carbon atom(s) in xcfx89-chain was modified have been known. But among such prostaglandins, there has been no publication which disclosed prostaglandins possessing unsubstituted or substituted phenyl in the xcfx89-chain of PG skeleton concretely.
For example, in Japanese Patent Application Kokai Sho 58-198466, it is disclosed that the following 5-thia-prostaglandin derivatives possess an activity of inhibition of blood plate aggregation. That is to say, it is disclosed that 5-thia-prostaglandins of the formula (A) 
(wherein, R1 is hydrogen or C1-10 alkyl,
R2 is substituted or unsubstituted C1-10 alkyl or substituted or unsubstituted C5xcx9c6 cycloalkyl,
R3 and R4 are, same or differently, hydrogen or protecting group)
or non-toxic salts thereof when R1 is hydrogen, possess an activity of inhibition of blood plate aggregation and a vasodilating activity, so they are useful as an agent for treatment or prevention of thrombosis and a hypertensive agent.
In this patent specification, the following xcfx89-cyclopentyl-compound is shown in Example 3 as a concrete compound: 
The present inventors have studied to find the stable compound which can bind to EP4 receptor specifically and which can bind to neither the other EP subtype receptors nor the other prostanoid receptors.
From the results, they have found that 5-thiaprostaglandins modified by introducing substituted phenyl intoxcfx89-chain of the said prostaglandins meet these purposes, and then have achieved the present invention.
As mentioned later, they have found that the prostaglandin compounds in which 5th carbon atom of xcex1-chain of PG skeleton is replaced with sulfur atom and in which phenyl substituted with a particular functional group is introduced into xcfx89-chain of PG skeleton can bind to EP4 strongly, bind to the other prostanoid receptors including the other subtype receptors weakly and are stable. And then, they have achieved the present invention.
The present invention relates to
(1) a 5-thia-xcfx89-substituted phenyl-prostaglandin E derivative of the formula (I) 
xe2x80x83(wherein, R1 is hydroxy, C1-6 alkyloxy or NR6R7 (in which R6 and R7 is, each independently, hydrogen or C1-4 alkyl),
R2 is oxo, halogen or Oxe2x80x94COR8 (in which R8 is C1-4 alkyl, phenyl or phenyl(C1-4 alkyl)),
R3 is hydrogen or hydroxy,
R4a and R4b is, each independently, hydrogen or C1-4 alkyl,
R5 is phenyl substituted with the following substituent(s):
i) 1xcx9c3 of
C1-4 alkyloxy-C1-4 alkyl,
C2-4 alkenyloxy-C1-4 alkyl,
C2-4 alkynyloxy-C1-4 alkyl,
C3-7 cycloalkyloxy-C1-4 alkyl,
C3-7 cycloalkyl(C1-4 alkyloxy)-C1-4 alkyl, phenyloxy-C1-4 alkyl,
phenyl-C1-4 alkyloxy-C1-4 alkyl,
C1-4 alkylthio-C1-4 alkyl,
C2-4 alkenylthio-C1-4 alkyl,
C2-4 alkynylthio-C1-4 alkyl,
C3-7 cycloalkylthio-C1-4 alkyl,
C3-7 cycloalkyl (C1-4 alkylthio)-C1-4 alkyl,
phenylthio-C1-4 alkyl or
phenyl-C1-4 alkylthio-C1-4 alkyl,
ii) C1-4 alkyloxy-C1-4 alkyl and C1-4 alkyl,
C1-4 alkyloxy-C1-4 alkyl and C1-4 alkyloxy,
C1-4 alkyloxy-C1-4 alkyl and hydroxy,
C1-4 alkyloxy-C1-4 alkyl and halogen,
C1-4 alkylthio-C1-4 alkyl and C1-4 alkyl,
C1-4 alkylthio-C1-4 alkyl and C1-4 alkyloxy,
C1-4 alkylthio-C1-4 alkyl and hydroxy or
C1-4 alkylthio-C1-4 alkyl and halogen,
iii) haloalkyl or hydroxy-C1-4 alkyl, or
iv) C1-4 alkyl and hydroxy;
{overscore (-----)} is single bond or double bond,
with the proviso that when R2 is Oxe2x80x94COR8, C8-C9 represents double bond) or a non-toxic salt thereof, or a cyclodextrin clathrate thereof,
(2) process for producing it, and
(3) a pharmaceutical composition comprising it as an active ingredient.
In the formula (I), C1-4 alkyl represented by R4a, R4b, R6, R7 and R8 and C1-4 alkyl in R5 and R8 means methyl, ethyl, propyl, butyl and isomers thereof.
In the formula (I), C1-6 alkyl represented by R1 means methyl, ethyl, propyl, butyl, pentyl, hexyl and isomers thereof.
In the formula (I), C2-4 alkenyl in R5 means vinyl, propenyl, butenyl and isomers thereof.
In the formula (I), C2-4 alkynyl in R5 means ethynyl, propynyl, butynyl and isomers thereof.
In the formula (I), C3-7 cycloalkyl in R5 means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
In the formula (I), halogen in R2 and R5 means fluoride, chloride, bromide and iodide.
In the present invention, the symbol  means single bond or double bond. Further, unless otherwise specified, in the present invention, the symbol  means that the substituent attached thereto is in front of the sheet, the symbol  means that the substituent attached thereto is behind the sheet and the symbol  or  means that there is a mixture of substituents in front of and behind the sheet or that the substituent attached thereto may be in front of or behind the sheet as would be clear to the person skilled in the art.
Unless otherwise specified, all isomers are included in the present invention. For example, alkyl, alkenyl, alkynyl, alkylene group means straight-chain or branched-chain ones. In addition, isomers on double bond, ring, fused ring (E-, Z-, cis-, trans-isomer), isomers generated from asymmetric carbon atom(s) (R-, S-, xcex1-, xcex2-isomer, enantiomer, diastereomer), optically active isomers (D-, L-, d-, I-isomer), polar compounds generated by chromatographic separation (more polar compound, less polar compound), equilibrium compounds, mixtures thereof at voluntary ratios and racemic mixtures are also included in the present invention.
In the formula (I), the substituent(s) of phenyl in R5 is/are preferably attached at 3-position, 3-position and 4-position, or 3-position and 5-position.
In the formula (I), each group (i) to (iv) as the substituent(s) of phenyl in R5 means as follows:
group i) means 1, 2 or 3 of alkyloxyalkyl etc.,
group ii) means at least one alkyloxyalkyl etc. and at least one alkyl, alkyloxy, hydroxy or halogen,
group iii) means alkyl substituted with 1 or 2 of halogen or hydroxy and
group iv) means at least one alkyl and at least one hydroxy.
Among the compounds of the present invention of the formula (I), the compounds described in the Examples, the compounds shown in the following Tables and the corresponding esters and amides are preferable.
[Salts]
The compounds of the present invention of the formula (I) may be converted into the corresponding salts by known methods. Non-toxic and water-soluble salts are preferable. Suitable salts, for example, are as follows: salts of alkali metals (potassium, sodium etc.), salts of alkaline earth metals (calcium, magnesium etc.), ammonium salts, salts of pharmaceutically acceptable organic amines (tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)aminomethane, lysine, arginine, N-methyl-D-glucamine etc.).
[Cyclodextrin Clathrate]
The compounds of the present invention of the formula (I) may be converted into the corresponding cyclodextrin clathrates by the method described in the specification of Japanese Patent Application Kokoku Sho 50-3362, 52-31404 or 61-52146 using xcex1-, xcex2- or xcex3-cyclodextrin or a mixture thereof. Converting into the corresponding cyclodextrin clathrates serves to increase the stability and solubility in water of the compounds, and therefore it is useful in the use for pharmaceuticals.
[Process for Producing the Compounds of the Present Invention]
(a) Compounds of the formula (I), wherein R1 is C1-6 alkyloxy i.e., the compounds of the formula (Ia) 
(wherein, R1-1 is C1-6 alkyloxy and the other symbols are as defined hereinbefore)
may be prepared from the compounds of the formula (II) 
(wherein, R3-1 is hydrogen or hydroxy protected by a protecting group which is removed under acidic conditions, R10 is a protecting group of hydroxy which is removed under acidic conditions, R5-1 is as defined for R5, provided that hydroxy group in R5-1 is protected by a protecting group which is removed under acidic conditions, and the other symbols are as defined hereinbefore) by the reaction for removal of a protecting group under acidic conditions.
The protecting group of hydroxy which is removed under acidic conditions includes, for example, t-butyidimethylsilyl, triphenylmethyl, tetrahydropyran-2-yl etc.
The hydrolysis under acidic conditions may be carried out by known methods. It may be carried out, for example, in an organic solvent miscible with water (e.g., tetrahydrofuran, methanol, ethanol, dimethoxyethane, acetonitrile or mixture thereof etc.) using an inorganic acid (e.g., hydrochloric acid, phospate, hydrofluoric acid, hydrogen fluoride-pyridine etc.), or an organic acid (e.g., acetic acid, tosylic acid, trichloroacetic acid etc.) at 0xcx9c50xc2x0 C.
(b) Compounds of the formula (I), wherein R1 is hydroxy i.e., the compounds of the formula (Ib) 
(wherein, all the symbols are as defined hereinbefore)
may be prepared from the compounds of the formula (Ia) 
xe2x80x83(wherein, all the symbols are as defined hereinbefore) by hydrogenolysis using an enzyme or by hydrogenolysis under alkaline conditions.
The hydrogenolysis using an enzyme may be carried out by known methods. It may be carried out, for example, in a mixed solvent of organic solvent miscible with water (e.g., ethanol, dimethylsulfoxide etc.) and water, in the presence or absence of buffer solution, using an enzyme for hydrogenolysis (esterase, lipase etc.) at 0xcx9c50xc2x0 C.
The hydrogenolysis under alkaline conditions may be carried out by known methods. It may be carried out, for example, in an organic solvent miscible with water (e.g., ethanol, tetrahydrofuran (THF), dioxane etc.) using an aqueous solution of alkali (sodium hydroxide, potassium hydroxide, potassium carbonate etc.) at xe2x88x9210xcx9c90xc2x0 C.
(c) Compounds of the formula (I), wherein R1 is NR6R7 i.e., the compounds of the formula (Ic) 
(wherein, all the symbols are as defined hereinbefore)
may be prepared by amidation of the compounds of the formula (Ib) 
xe2x80x83(wherein, all the symbols are as defined hereinbefore)
with the compounds of the formula (III)
HNR6R7xe2x80x83xe2x80x83(III)
(wherein, all the symbols are as defined hereinbefore).
Amidation may be carried out by known methods. For example, it may be carried out in an inert organic solvent (THF, methylene chloride, benzene, acetone, acetonitrile or mixture thereof etc.), in the presence or absence of a tertiary amine (dimethylaminopyridine, pyridine, triethylamine etc.), using a condensing agent (1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) etc.) at 0xcx9c50xc2x0 C.
The compounds of the formula (III) have been known or may be prepared by known methods easily.
The compounds of the formula (II) may be prepared by the following Reaction Schemes 1-5.
In each Reaction Scheme, the symbols are as defined hereinbefore or are as follows:
t-Bu: t-butyl,
Et: ethyl,
Ms: methanesulfonyl,
DMAP: dimethylaminopyridine,
n-Bu: normal butyl,
AIBN: 2,2xe2x80x2-azobisisobutyronitrile,
Ts: p-toluenesulfonyl,
R2-1: halogen,
Ac: acetyl,
TMS: trimethylsilyl. 
[Starting Materials and Reagents]
Each Reaction in the said Reaction Schemes may be carried out by known methods. In the said Reaction Schemes, the compounds of the formulae (IV), (V), (XVI), (XIV), (VI) and (XXIII) as starting materials have been known or may be prepared easily by known methods.
For example, the compounds of the formula (XIV) wherein R3-1 is THP have been described in J. Am. Chem. Soc., 98, 1490 (1971).
The other starting materials and reagents in the present invention are known per se or may be prepared by known methods.
In each reaction in the present specification, reaction products may be purified by conventional techniques. For example, purification may be carried out by distillation at atmospheric or reduced pressure, by high performance liquid chromatography, by thin layer chromatography or by column chromatography using silica gel or magnesium silicate, by washing or by recrystallization. Purification may be carried out after each reaction, or after a series of reactions.
[Pharmacological Activities]
The compounds of the present invention of the formula (I) can bind strongly and show an activity on the EP4 subtype receptor which is one of the PGE2 receptors.
For example, in a standard laboratory test, such effects of the compound of the present invention were confirmed by binding assay using cell expressing the prostanoid receptor subtypes. (i) Binding assay using cell expressing the prostanoid receptor subtypes The preparation of membrane fraction was carried out according to the method of Sugimoto et al (J. Biol. Chem., 267, 6463-6466 (1992)), using CHO cell expressing prostanoid receptor subtype (mouse EP1, EP2, EP3, and EP4, and human IP).
The reaction solution (200 xcexcl) containing membrane fraction (0.5 mg/ml), [3H]-PGE2 was incubated for 1 hour at room temperature. The reaction was terminated by addition of 3 ml of ice-cold buffer. The mixture was rapidly filtered under reduced pressure through a glass filter (GF/B). The radioactivity associated with the filter was measured by liquid scintillation counter.
Kd and Bmax values were determined from Scatchard plots [Ann. N.Y. Acad. Sci., 51, 660 (1949)]. Non-specific binding was calculated as the amount bound in the presence of an excess (2.5 xcexcM) of unlabeled PGE2. In the experiment for competition of specific [3H]-PGE2 binding by the compounds of the present invention, [3H]-PGE2 was added at a concentration of 2.5 nM and the compound of the present invention was added at various concentrations. The following buffer was used in all reactions. Buffer: 10 mM potassium phosphate (pH6.0), 1 mM EDTA, 10 mM MgCl2, 0.1 M NaCl
The dissociation constant Ki (xcexcM) of each compound was calculated by the following equation.
Ki=IC50/(1+([C]/Kd))
The results are shown in Table 4.
As shown in the above results, the compounds of the present invention can bind to receptor for subtype EP4 strongly and do not bind to other receptors for PGE2.
[Toxicity]
The toxicity of the compounds of the formula (I) of the present invention is very low and therefore, it is confirmed that these compounds are safe for use as medicine. For example, the maximum tolerance dose of the compound of Example 1 by i.v. route in rat was 30 mg/kg body weight or more.
The compounds of the present invention of the formula (I) can bind and show the activity on the PGE2 receptor. Particularly, they bind EP4 subtype receptor strongly, so they are useful for the prevention and/or treatment of immunological diseases (autoimmune diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis, Sjoegren""s syndrome, chronic rheumarthrosis and systemic lupus erythematosus etc., and rejection after organ transplantation etc.), asthma, abnormal bone formation, neuronal cell death, lung failure, liver damage, acute hepatitis, nephritis, renal insufficiency, hypertension, myocardiac ischemia, systemic inflammatory response syndrome, ambustion pain, sepsis, hemophagous syndrome, macrophage activation syndrome, Still""s disease, Kawasaki disease, burn, systemic granulomatosis, ulcerative colitis, Crohn""s disease, hypercytokinemia at dialysis, multiple organ failure, and shock etc. Further, it is thought that EP4 subtype receptor relates to sleeping disorder and blood platelet aggregation, so the compounds of the present invention are expected to be useful for the prevention and/or treatment of such diseases.
The compounds of the present invention of the formula (I) bind weakly to the other subtype receptors and do not express other effects, therefore such compounds are expected to be an agent having less side effect.
For the purpose above described, the compounds of the formula (I) of the present invention, non-toxic salts thereof, or cyclodextrin clathrates thereof may be normally administered systemically or locally, usually by oral or parenteral administration (administration into joint or subcutaneous administration etc. is included).
The doses to be administered are determined depending upon age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment etc. In the human adult, the doses per person per dose are generally between 1 xcexcg and 100 mg, by oral administration, up to several times per day, and between 0.1 xcexcg and 10 mg, by parenteral administration (preferred into vein) up to several times per day, or continuous administration between 1 and 24 hrs. per day into vein.
As mentioned above, the doses to be used depend upon various conditions. Therefore, there are cases in which doses lower than or greater than the ranges specified above may be used.
The compounds of the present invention may be administered as solid compositions, liquid compositions or other compositions for oral administration, or as injections, liniments or suppositories etc. for parenteral administration.
Solid compositions for oral administration include compressed tablets, pills, capsules, dispersible powders, and granules.
Capsules contain hard capsules and soft capsules.
In such compositions, one or more of the active compound(s) is or are, admixed with at least one inert diluent such as lactose, mannitol, mannit, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium metasilicate aluminate.
The compositions may also comprise, as is normal practice, additional substances other than inert diluents: e.g. lubricating agents such as magnesium stearate, disintegrating agents such as cellulose calcium glycolate, and agents to assist dissolution such as glutamic acid, asparaginic acid. The tablets or pills may, if desired, be coated with film of gastric or enteric material such as sugar, gelatin, hydroxypropyl cellulose or hydroxypropyl cellulose phthalate etc., or be coated with two or more films. And further, coating may include containment within capsules of absorbable materials such as gelatin.
Liquid compositions for oral administration include pharmaceutically-acceptable emulsions, solutions, syrups and elixirs etc. In such liquid compositions, one or more of the active compound(s) is or are comprised in inert diluent(s) commonly used in the art (for example, purified water, ethanol etc.). Besides inert diluents, such compositions may also comprise adjuvants such as wetting agents, suspending agents, sweetening agents, flavouring agents, perfuming agents and preserving agents.
Other compositions for oral administration include spray compositions which may be prepared by known methods and which comprise one or more of the active compound(s). Spray compositions may comprise additional substances other than inert diluents: e.g. stabilizing agents such as sodium hydrogen sulfate, stabilizing agents to give isotonicity, isotonic buffer such as sodium chloride, sodium citrate, citric acid. For preparation of such spray compositions, for example, the method described in the U.S. Pat. Nos. 2,868,691 or 3,095,355 may be used.
Injections for parenteral administration include ste rile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solutions or suspensions include distilled water for injection and physiological salt solution. Non-aqueous solutions or suspensions include propylene glycol, polyethylene glycol, plant oil such as olive oil, alcohol such as ethanol, POLYSORBATE80 (registered trade mark) etc.
Such compositions may comprise additional diluents: e.g. preserving agents, wetting agents, emulsifying agents, dispersing agents, stabilizing agent, assisting agents such as agents to assist dissolution (for example, glutamic acid, asparaginic acid). They may be sterilized for example, by filtration through a bacteria-retaining filter, by incorporation of sterilizing agents in the compositions or by irradiation. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile diluent for injection immediately before use.
Other compositions for parenteral administration include liquids for external use, and endermic liniments, ointments, suppositories and pessaries which comprise one or more of the active compound(s) and may be prepared by known methods.